JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1989, p. 1997-20020095-1137/89/091997-06$02.00/0Copyright © 1989, American Society for Microbiology

Stool Diagnosis of Giardiasis Using a Commercially AvailableEnzyme Immunoassay To Detect Giardia-Specific

Antigen 65 (GSA 65)JOHN D. ROSOFF,1* CYNTHIA A. SANDERS,2 SEEMA S. SONNAD,' PAUL R. DE LAY,3 W. KEITH HADLEY,2

FRANK F. VINCENZI,4 DAVID M. YAJKO,2 AND PETER D. O'HANLEY'

Departments of Medicine (Infectious Disease) and Medical Microbiology, Stanford University, Stanford, California943051; Refugee Screening Clinic3 and Department of Laboratory Medicine,2 University of California, San FranciscoGeneral Hospital, San Francisco, California 94110; and Department ofPharmacology, University of Washington,

Seattle, Washington 981954

Received 27 March 1989/Accepted 8 June 1989

A commercially available enzyme immunoassay for the diagnosis of giardiasis was evaluated in a clinicaltrial. The ProSpecT/Giardia diagnostic test (Alexon, Inc., Mountain View, Calif.) was compared with thestandard ova and parasite (O&P) microscopic examination. Additionally, several widely used stool fixativesand a commonly used transport medium were assessed for compatibility with the immunoassay. A total of 325stool specimens were collected and used to evaluate assay performance. Of those, 93 specimens were collectedfrom symptomatic Giardia O&P-positive patients and 232 specimens were randomly collected from patients aspart of a routine health screening procedure. All 93 Giardia O&P-positive stool specimens were stronglypositive by visual and spectrophotometric examination using the immunoassay. Of the 232 randomly collectedspecimens, 16 were positive by O&P examination and immunoassay, 6 were negative by O&P examination butpositive by immunoassay, and was positive by O&P examination and negative by immunoassay. There wassubstantial supportive evidence that indicated that the six immunoassay-positive, O&P-negative specimenswere true-positives. When these six specimens were accepted as true-positives, the immunoassay detectedalmost 30% more cases of Giardia infection than did O&P examination. Its sensitivity and specificity were 96and 100%, respectively, while the sensitivity and specificity of O&P examination were 74 and 100%,respectively. The immunoassay also performed well on specimens treated with 10% neutral Formalin, sodiumacetate-Formalin fixative, and Cary-Blair transport medium. However, the test was not compatible withpolyvinyl alcohol-treated specimens. Overall, the ProSpecT/Giardia test was a sensitive, specific immunoassaywhich was easy to run and interpret. It offers a simple solution to traditional difficulties encountered indiagnosing Giardia infection.

Diagnosis of Giardia lamblia infection by microscopicexamination of stool for ova and parasites (O&P) is alaborious process (6, 7). Iodine-stained wet smears, tri-chrome-stained cyst concentrates prepared by Formalin-ethyl acetate centrifugation or by zinc sulfate flotation, andtrichrome-stained polyvinyl alcohol (PVA)-preserved stoolsare standard methods of stool preparation used to increasethe sensitivity of Giardia detection (3, 12, 26). Even afterapplication of these techniques, the sensitivity of O&Pmicroscopic examination is dependent upon the skill of themicroscopist and the time spent scanning each preparation.Clinicians agree that the diagnostic success rate of stoolexamination in most clinical laboratories is roughly 50 to70% (2, 3, 12, 14). To further complicate matters, manypatients with Giardia infection excrete infectious cysts instool intermittently (14), necessitating collection and exam-ination of several stools from a patient over several days toweeks.

In recent years, efforts have been made to improve thesensitivity of Giardia diagnosis. These efforts have focusedprimarily on serologic testing for anti-Giardia antibodies (8,11, 20, 23, 25) and detection of Giardia antigens in patientstool specimens (5, 9, 22, 24). Serologic tests have proven tobe of little value in Giardia diagnosis because there is littlecorrelation between positive anti-Giardia antibody titers and

* Corresponding author.

the presence of active Giardia infection. Cross-reactionswith microbial antigens have also caused problems (10).Stool antigen detection tests have enjoyed greater successand appear to correlate well with current infection. Potentialproblems with stool antigen detection tests are as follows. (i)They usually use polyspecific polyclonal antibodies againstthe whole G. lamblia trophozoite and/or cyst, raising con-cerns of nonspecific reactions or of cross-reactivity withother gastrointestinal parasites (28). (ii) Most antigens tar-geted in these polyvalent polyspecific antibody-based assaysare labile in conventional laboratory fixatives and mediaused for collection, transport, and storage of stool specimensdestined for O&P microscopic examination or stool culture(17). These immunoassays demand the use of untreated stoolspecimens and impose limitations on existing stool collectionprocedures. Recently, we reported the isolation of a Giardia-specific stool antigen (GSA 65) useful in the diagnosis ofgiardiasis (17). GSA 65 possesses the qualities of a diagnos-tically ideal stool antigen. (i) It is specific to G. lamblia. (ii)It is stable in the host gastrointestinal tract. (iii) It is stable instandard laboratory fixatives. (iv) It is present in immuno-logically detectable quantities (18). Moreover, GSA 65 maybe the most abundant immunologically detectable Giardiaantigen in the stool of giardiasis patients (17). We previouslydesigned and successfully tested an experimental antigencapture enzyme-linked immunosorbent assay (ELISA) todetect GSA 65 in aqueous stool eluates of giardiasis patients

1997

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(J. D. Rosoff, C. A. Sanders, P. R. De Lay, W. K. Hadley,D. M. Yajko, and P. H. O'Hanley, Program Abstr. 28thIntersci. Conf. Antimicrob. Agents Chemother., abstr. no.402, 1988). We now report performance data on a newcommercially available coproimmunodiagnostic test for giar-diasis (ProSpecT/Giardia; Alexon, Inc., Mountain View,Calif.) that uses monospecific anti-GSA 65 antibodies.

MATERIALS AND METHODS

Clinical facilities used for stool specimen collection. Stoolspecimens used in the ProSpecTlGiardia diagnostic teststudy were collected from four sources: (i) Sacred Heart and(ii) Holy Family Hospitals in Spokane, Wash. (iii) theRefugee Screening Clinic at San Francisco General Hospital,San Francisco, Calif.; and (iv) the parasitology division ofthe clinical microbiology facility at San Francisco GeneralHospital. Permission for specimen collection and disclosureof confidential patient information was obtained at eachfacility via clinicians and researchers in accordance withguidelines established by the Human Subjects Committees atthe various institutions.Specimen collection methods differed at the Spokane and

the San Francisco facilities. Spokane facilities collectedstool specimens only from patients with gastrointestinalcomplaints. For this reason, the Sacred Heart and HolyFamily Hospitals were used as a source of Giardia O&P-positive stool specimens only; no negative control speci-mens were collected at these facilities. The Refugee Screen-ing Clinic in San Francisco collected stool specimens as partof a routine health screening procedure that every newpatient undergoes on entry to the United States. A survey ofthis patient population has shown a high prevalence ofGiardia infection (1). The unbiased manner of specimencollection, in conjunction with the high prevalence of giar-diasis, made the Refugee Screening Clinic an ideal facilityfor conducting a clinical trial to evaluate the utility of theimmunoassay. Both Giardia-positive and Giardia-negativestool specimens were collected at this facility. In addition tothe first three sources, stool specimens containing parasitesother than G. lamblia by O&P examination were collectedfrom the parasitology division of the clinical microbiologyfacility at San Francisco General Hospital. These specimenswere grouped with specimens from the Refugee ScreeningClinic that were O&P positive for intestinal parasites otherthan G. lamblia to make up a cross-reactivity test panel forthe immunoassay.

Stool specimen and patient data collection. Single speci-mens from each patient were initially collected in the un-treated state. At the Spokane facilities, equal samples fromspecimens submitted to the clinical microbiology laborato-ries were concentrated by the Formalin-ethyl acetate orFormalin-ether method, and iodine-stained wet mounts weremicroscopically examined for cysts within 1 day of specimensubmission. Trichrome stains of nonconcentrated smearswere also microscopically examined for the presence oftrophozoites (2, 3, 26). Giardia O&P-positive specimenswere stored refrigerated (4°C) for 2 to 8 weeks at eachclinical facility until they were picked up. They were subse-quently frozen at -20°C until used. Specimens from theRefugee Screening Clinic in San Francisco were collected ina blinded fashion. At the end of each working day, patientspecimens were picked up from the clinic and equal samplesof stool were removed, labeled with study identificationnumbers, and frozen. The remainder of the specimens werereturned to the parasitology laboratory for O&P microscopic

examination using the methods cited above. All new patientswere automatically entered into the Giardia diagnostic studyat the Refugee Screening Clinic. The same collection proto-cols were followed on the cross-reactivity test panel stoolspecimens obtained from the parasitology laboratory at theSan Francisco General Hospital.At each facility, the patient data collected were similar.

Patient name, gender, and date of birth, accession numberand/or patient identification number, quantity and types ofparasites found on O&P examination, collection facilityname, and special comments were entered into a data base.Additionally, stool consistency data on specimens fromSpokane were collected. All data collected as part of therefugee clinic study were entered into the data base by aninvestigator who had no knowledge of the immunoassayresults.

Compatibility assessment of fixatives and transport mediumwith the immunoassay. The compatibility of the immunoas-say with commonly used stool fixatives, as well as with acommon transport medium, was assessed. The pH of theTrizma specimen dilution buffer at a 1:20 (vol/vol) ratio(following the immunoassay directions) of fixative to bufferwas determined by using sodium acetate-Formalin (SAF) orPVA fixative, 10% Formalin, Cary-Blair transport medium,and distilled water as a control.The effects of fixatives and a transport medium on the

immunoassay sensitivity were assessed. Thirty randomlyselected frozen specimens from the Giardia O&P-positivespecimen collection were diluted 1:4 (vol/vol) in 10% For-malin or SAF fixative, in Cary-Blair transport medium, or inphosphate-buffered saline (PBS) (pH 7.4, 35 mM) containing0.05% Tween 20 (PBS-T) as a control. Specimens treatedwith 10% Formalin or SAF fixative were stored for 1 monthat ambient temperature (20 to 25°C). Specimens treated withCary-Blair medium or PBS-T were stored for 1 month at 4°C.All treated specimens were subsequently diluted 1:20 inTrizma specimen dilution buffer and assayed without cen-trifugation or gravity settling of the specimen.

Titration experiments on five treated Giardia O&P-posi-tive specimens and five negative control specimens wereconducted to ascertain the specimen dilution at which visualpositivity becomes difficult to judge with the immunoassay.Specimens in Cary-Blair transport medium, 10% Formalin,SAF fixative, or PBS-T were diluted 1:20, 1:200, 1:2,000,1:20,000 and 1:200,000 by serial dilution in Trizma specimendilution buffer and assayed as described below.Specimen preparation and assay. Treated and untreated

specimens were prepared and assayed at a Stanford labora-tory removed from the clinical trial location following theProSpecT/Giardia directions. Patient stool specimens werediluted (1:20 [vol/vol]) in Trizma-based specimen dilutionbuffer to create a stool eluate. Specimen eluates (200 iil)were added to antibody-coated assay tubes in duplicate andincubated for 1 h at room temperature. The tubes werewashed three times with phosphate-based wash buffer using3 ml per wash. Enzyme conjugate (200 ,uI) was added to eachimmunoassay tube and incubated for 1 h at room tempera-ture. The tubes were washed four times using 3 ml of washbuffer per wash and immediately adding 200 ,ul of o-phe-nylenediamine-peroxide buffer substrate solution. After 10min, reactions were stopped with 50 ,ul of 2 N sulfuric acidsolution, and the reaction was read visually. The test wasread visually following the directions and using the assayinterpretation card included with each ProSpecT/Giardia kit.Any detectable color generation was regarded as a positiveassay outcome. Following visual assessment of positivity or

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TABLE 1. Comparison of Giardia cyst and trophozoite countsobserved by O&P examination of fecal concentrates andimmunoassay ODs of Giardia-positive specimens from the

Spokane and San Francisco study populations

Spokane San FranciscoMicroscopic quantity specimens (n = 93) specimens (n = 23)of Giardia cysts andtrophozoites on O&P Avg Avg

examination No. immunoassay No. immunoassayOD OD

None 1 2.000 6 1.608Rare 2 2.000 4 0.525Occasional NAb NAb 1 1.234Few 25 2.000 4 1.300Moderate 40 1.930 5 1.698Abundant 25 1.922 3 1.500

a Counts per 22-mm cover slip: rare, 1 to 5; occasional, 6 to 10; few, 11 to20 (for Spokane, 6 to 20); moderate, 21 to 40; abundant, >40.

b NA, Not applicable. Spokane facilities did not use "occasional" in theirparasite count protocol.

negativity, the contents of the tubes were transferred to96-well polystyrene plates and spectrophotometrically readat A490 on an MR 580 MicroElisa plate reader (DynatechLaboratories, Inc., Chantilly, Va.). Spectrophotometric dataare presented as a means to minimize subjectivity of inter-pretation during this study as well as to provide quantitativemeasurement of exactly how much color development therewas for each reaction.

RESULTS

Sensitivity and specificity of the immunoassay. Initially thesensitivity of the immunoassay was tested using 93 G.lamblia O&P-positive clinical specimens collected at theHoly Family and Sacred Heart Hospitals from July 1986 toAugust 1987. All 93 of these specimens were stronglypositive by visual examination using the immunoassay. Ofthese specimens, 87 had optical densities (ODs) greater than2.00, which was off-scale on the spectrophotometer. Theremaining six had ODs greater than 1.00. The eye can easilydetect down to 0.40 OD in the yellow range. The average ODof the 93 specimens was 1.95. There was no correlationbetween the quantity of cysts or trophozoites present instools of various consistencies as assessed by O&P exami-nation and the final OD generated by the immunoassay(Table 1). Four negative control stool specimens (two ofwhich were run in duplicate) were visually devoid of colorfollowing the assay. The optical density mean and range ofthese specimens were 0.164 and 0.01, respectively. Thesespecimens were collected following the same proceduresused at San Francisco General Hospital.These results led us to evaluate the utility of the immu-

noassay to detect Giardia infection in symptomatic andasymptomatic patients in a clinical trial at the RefugeeScreening Clinic. From August to October 1987, 208 patientstool specimens were collected. Of these specimens, 16 werepositive by O&P examination and by immunoassay, 6 werenegative by O&P examination and positive by immunoassay,and 1 was positive by O&P examination and negative byimmunoassay. The average OD of the 22 specimens judgedvisually positive was 1.36; 8 had ODs greater than 2.00, 10had ODs greater than 1.00, and the remaining 4 had ODsranging from 0.474 to 0.659. The mean OD and standarddeviation of the 186 specimens judged as visually negativewas 0.180 + 0.04. Similar to the Spokane study specimens,

TABLE 2. List of all parasites, other than G. lamblia,represented in the cross-reactivity test panel

No. of No. of immunoassay-Intestinal parasite specimens positive reactionsofal

withprasite specimens tested inplthparasite each category

Ascaris lumbricoides 9 1Blastocystis hominis 3 0Chilomastix mesnili 2 0Clonorchis sinensis 1 0Cryptosporidium sp. 4 0Entamoeba coli 9 1Entamoeba hartmanni 2 0Entamoeba histolytica 3 0Endolimax nana 8 0Hookworm 8 0Hymenolepsis nana 2 0Iodamoeba buetschlii 6 0Isospora belli 1 0Strongyloides stercoralis 1 0Trichuris trichiura 8 0

a These specimens were negative for G. lamblia by O&P microscopicexamination.

there was a poor correlation between the microscopicallyobserved cyst or trophozoite count and the intensity ofimmunoassay color development (Table 1).

In addition, 51 stool specimens containing 15 intestinalparasites other than G. lamblia by O&P examination werecollected at San Francisco General Hospital from August toOctober 1987. These specimens were used for cross-reac-tivity testing of the immunoassay (Table 2). Of these speci-mens, 27 were collected from the Refugee Screening Clinicand 24 were collected from patients seen at other clinics. Ofthe 51 specimens collected, only two positive reactions wereobserved. The assay ODs of these two specimens were 1.128and 1.157, respectively. The OD mean and standard devia-tion of the remaining 49 negative specimens were 0.181 ±0.036.

Compatibility of fixatives and a medium with the immunoas-say. The ability of the Trizma specimen dilution buffer tostabilize several common stool fixatives and a stool transportmedium was used to determine the compatibilities of theseagents with the immunoassay. Each agent was consideredcompatible if its postdilution pH was .7.0. At this pH,antigen-antibody interactions critical to the outcome of apositive diagnostic test occur. When SAF fixative, 10%Formalin, and Cary-Blair medium were diluted in Trizmaspecimen dilution buffer, they produced postdilution pHs of7.0 to 7.5. These agents were compatible with the immunoas-say. The postdilution pH of PVA was low (6.0 to 6.5). Thisfixative was incompatible with the immunoassay.Immunoassay sensitivity using treated specimens. Thirty

Giardia O&P-positive stool specimens were each treatedwith 10% Formalin, SAF fixative, Cary-Blair transport me-dium, or PBS-T and evaluated by the immunoassay. Treat-ment of stools with any of these agents did not significantlyalter the immunoassay sensitivity. The means and standarddeviations of the 30 cohort specimens in each agent treat-ment category were as follows: PBS-T, 1.730 + 0.449;Cary-Blair medium, 1.667 + 0.478; SAF fixative, 1.658 +

0.497; and 10% Formalin, 1.556 + 0.495. The six negativecontrols did not develop observable color and had an aver-age OD of 0.164 (range, + 0.010). Figure 1 shows theaveraged titration data from five Giardia O&P-positive stoolspecimens treated with the agents listed above and five

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2

01'

1

0.4

01 2 3 4

0.5 x -log DILUTIONReromended specimen dilutionforthe ProSpet TlGiardiaT" test (1:20).

FIG. 1. Averaged titration data from five Giardia-positive stoolspecimens treated with PBS-T (positive control), Cary-Blair me-dium, 10% Formalin, or SAF fixative and five untreated Giardia-negative control stool specimens. All specimens were stored for 1month in their respective fixatives prior to assay. Note that assayODs decrease as a function of decreasing GSA 65 concentration.

negative controls. There was negligible loss in assay sensi-tivity when specimens were treated with Cary-Blair trans-port medium, SAF fixative, or 10% Formalin. Positivity wasvisually detected at specimen dilutions in excess of 1:1,000in all treatment categories.

DISCUSSION

A new immunoassay for the diagnosis of Giardia infectionhas been clinically tested. This immunoassay is based on thedetection of Giardia-specific antigen 65 (GSA 65) in aqueousextracts of stool specimens with monospecific polyclonalsera (17, 18).The ProSpecTiGiardia test was sensitive and specific. The

test was read visually, according to the directions included inthe assay kit, and spectrophotometrically to provide anobjective means for comparison of specimen-specific testoutcomes. Experimental results showed that the immunoas-say was more sensitive than O&P examination and routinelyproduced outcomes that were easy to interpret. Specimensthat produced test outcomes which generated color wereinterpreted as positive; those with no visually discerniblecolor development were interpreted as negative. No ambig-uous colorometric outcomes were encountered.

All 93 known-positive specimens collected from Spokaneclinical facilities were strongly positive by the immunoassayregardless of O&P cyst and trophozoite counts (Table 1) orstool consistency. All specimens reported as having fewcysts and/or trophozoites on microscopic examination werestrongly positive, with ODs usually off-scale spectrophoto-metrically (OD, >2.00). One stool specimen which con-tained no Giardia cysts or trophozoites by O&P examinationalso had an OD of >2.00 by the immunoassay. This speci-

men was collected from a patient with unexplained gastro-intestinal distress who submitted a second stool specimen 2days later that was O&P Giardia positive. All of the speci-mens which yielded lower assay ODs (1.374, 1.445, 1.007,1.445, 1.166, and 1.163) contained moderate or abundantnumbers of Giardia cysts and/or trophozoites. This inverserelationship between the number of cysts and trophozoitesexcreted and the density of colorometric development by theimmunoassay was opposite to the expected results. A similarinverse relationship was found in the immunoassay-positivespecimens from the San Francisco clinical population. How-ever, the average OD generated by the immunoassay-posi-tive specimens from the San Francisco Refugee Clinic wassignificantly lower than the average OD generated by Spo-kane specimens (1.36 versus >1.95). Two possible explana-tions for this discrepancy exist. The first is that althoughstool specimens from both study populations were collectedsimilarly, they were treated differently after collection, re-sulting in more free Giardia antigen being shed into the stool.San Francisco Refugee Clinic patient specimens were frozenimmediately following submission. Spokane patient speci-mens were stored refrigerated for prolonged periods beforethey were frozen. It is well known that Giardia cysts inuntreated stool degenerate rapidly when stored refrigeratedfor more than a few days. GSA 65 may have been a majorcyst breakdown product released into the stool specimenscollected in Spokane. One may then speculate that sincespecimens collected in San Francisco were frozen immedi-ately, little or no cyst breakdown occurred and GSA 65levels remained stable. It should be mentioned, however,that in subsequent studies that we have conducted, GSA 65appears to decrease rather than increase in untreated stoolspecimens stored at 4°C over the same time period. Thesecond possible explanation is that the difference in ODgeneration by specimens from the two study populations ispossibly the result of a difference between the two popula-tions. Specimens collected in Spokane came from patientswith gastrointestinal disturbances. Specimens from the SanFrancisco Refugee Screening Clinic were submitted as partof a routine health screening procedure. Most of thesepatients did not have gastrointestinal problems. From thesedata it may be speculated that more GSA 65 is shed into thestool of symptomatic or acute giardiasis patients than ofthose who are asymptomatic carriers or have chronic infec-tions. If this is true, then the colorometric density generatedby immunoassay, which is proportional to the concentrationof GSA 65 present in stool (Fig. 1), correlates positively withpatient symptoms. Efforts are currently under way to deter-mine if these findings are reproducible in other study popu-lations.Of the 22 immunoassay-positive specimens from San

Francisco, 6 were O&P negative. All six of the assayoutcomes for these specimens were distinctly positive andoptically dense (2.000, 1.128, 1.364, 2.000, 1.157, and 2.000).Four of these patients had immediate family members withconcurrent Giardia infection (67% as compared with 7% of arandom sample [n = 42] of patients with assay-negative,O&P-negative specimens). Another of these patients camefrom a family in which four members had concurrent infec-tion with several other intestinal parasites. While the sixthpatient did not have family members with Giardia or otherintestinal parasitic infections, she did have unexplainedintermittent gastrointestinal distress. All of these patientswere from Southeast Asia, a geographical region wheregiardiasis is prevalent. If the six assay-positive, O&P nega-tive specimens are assumed to be positive, the percent

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Giardia-positive outcomes for Southeast Asian patients is11.4%. This number is probably more realistic for this regionthan the 8% obtained when these six specimens are consid-ered negative. For these reasons, all six immunoassay-positive, O&P-negative specimens were accepted as true-positives missed by O&P microscopic examination.When O&P examination was accepted as the "gold stan-

dard" (100% sensitivity, 100% specificity), the immunoassaywas 94% sensitive and 97% specific. When the six immu-noassay-positive, O&P-negative specimens were included astrue-positives, the respective sensitivity and specificity ofthe immunoassay rose to 96 and 100%, while O&P sensitiv-ity dropped to 74%. The immunoassay detected almost 30%more cases of giardiasis than did O&P examination.The immunoassay was also highly specific when tested on

stool specimens containing 15 common intestinal parasitesother than G. lamblia. The two positive reactions recordedin the cross-reactivity test panel came from Refugee Screen-ing Clinic patients who had young immediate family mem-bers with concurrent Giardia infection. These reactionsmost likely represent cryptic cases of giardiasis rather thantrue cross-reactions due to lack of reactivity with severalother specimens in the same parasite category. A highperson-to-person transmission rate within the family settingis widely acknowledged to occur in cases of Giardia infec-tion, particularly transmission from infants and children toadults (4, 15, 20).

Several stool antigen detection ELISAs for Giardia spp.have been reported (13, 16, 21). All of these assays havebeen designed and used for research purposes and demandthe use of expensive spectrophotometric equipment to de-termine assay results. These tests have used polyclonalpolyspecific anti-trophozoite and/or anti-cyst sera to detect abroad array of Giardia antigens. Janoff et al. (13) comparedELISA and O&P examination and concluded that ELISAwas a sensitive and specific diagnostic tool. On the basis oftheir data, they felt that ELISA might be more sensitive thanmicroscopy. Nash et al. (16) used an antigen detectionELISA to monitor known infected and noninfected patients.They found that ELISA was at least as good at distinguishingknown infected and noninfected patients as extremely dili-gent O&P tests. Their conclusion was that ELISA wasprobably much better than an average clinical examination.

ProSpecT!Giardia is the first commercially available anti-gen detection ELISA for Giardia diagnostic testing. This testis unlike previously reported assays in that it detects a singleGiardia stool antigen. Building a diagnostic test around asingle defined antigen improves test specificity. The poten-tial for cross-reactions is drastically reduced when antibod-ies are directed against a few defined epitopes rather than atthousands of undefined epitopes. Cross-reactivity test re-sults from this research and our previous research (17, 18)support this conclusion. The ProSpecT/Giardia test rou-tinely produced assay outcomes much more optically densethan those reported previously. This eliminates the need forspectrophotometric equipment and results in a test which isvisually read and easily interpreted. The ability of theProSpecTiGiardia test to function with a variety of stoolfixatives, as well as with Cary-Blair transport medium, isclinically advantageous. This test accommodates traditionalstool collection with the exception of collection in PVAfixative. As a result, the immunoassay requires minimalchanges in routine collection techniques. The stability ofGSA 65 offers flexibility in the choice of fixatives used forstool collection.GSA 65 is stable in fixative-treated stools and untreated

stools for long periods (17, 18; Rosoff et al., 28th ICAAC).Data presented in this paper show that there is minimaldecrease in assay OD generation when Giardia-positivestool specimens are stored in conventional fixatives over a1-month period (Fig. 1). Giardia-positive stool specimensfixed in 10% Formalin or SAF fixative and stored at roomtemperature for longer than 6 months have been observed toremain assay positive (data available on request). Untreatedfrozen Giardia-positive stool specimens and specimensstored frozen in Cary-Blair transport medium retain theirassay positivity for at least 1 year (data available on request).These findings suggest that the ProSpecT/Giardia test is notonly practical as a diagnostic tool but also useful in epide-miologic surveys in which collected specimens may sit formany months prior to examination.The ProSpecT/Giardia immunoassay offers several advan-

tages over O&P examination. It is not labor intensive;incubations aside, labor time involved in running the test isless than 10 min. The time investment involved in running 1test versus 10 is small; additional time is invested in prepar-ing the extra specimens prior to assay initiation. Testing caneasily be performed on batches of specimens. The assay iseasy to run, and the interpretation is straightforward.

This immunoassay is not a substitute for O&P examina-tion. The usefulness of O&P examination is due not to itssensitivity but to its ability to broadly screen for infectionwith virtually any intestinal parasite. With several knownhuman intestinal parasites, it is unlikely that a single diag-nostic test will replace O&P examination in the near future,although this is a long-term goal in this laboratory. Thisclinical trial shows that the ProSpecT!Giardia immunoassaycan rule out infection with G. lamblia, a major cause ofdiarrheal disease. If the data presented in this paper arerepresentative of a larger clinical sample, this immunoassaycan detect Giardia infection in at least 30% more cases thanmicroscopic examination. This large percentage of previ-ously undetected cases suggests that the test should be run inconjunction with O&P microscopic examination.

ACKNOWLEDGMENTS

We express our sincere thanks to the following people who havemade this study possible: the parasitology and Refugee ScreeningClinic staffs at San Francisco General Hospital, Margaret Hold-ridge, Don Anderson, Jerry Claridge, Karen Olsen, Larry Bernard,and the Sacred Heart and Holy Family Hospital Clinical Microbiol-ogy staffs in Spokane, Wash.

This research was supported, in part, by Rockefeller Foundationgrant GA HS 8713.

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